cDNA-AFLP analysis reveals the adaptive responses of citrus to long-term boron-toxicity

Boron (B)-toxicity is an important disorder in agricultural regions across the world. Seedlings of ‘Sour pummelo’ (Citrus grandis) and ‘Xuegan’ (Citrus sinensis) were fertigated every other day until drip with 10 μM (control) or 400 μM (B-toxic) H3BO3 in a complete nutrient solution for 15 weeks.

Guo et al BMC Plant Biology 2014, 14:284 http://www.biomedcentral.com/1471-2229/14/284 RESEARCH ARTICLE Open Access cDNA-AFLP analysis reveals the adaptive responses of citrus to long-term boron-toxicity Peng Guo1,2, Yi-Ping Qi3, Lin-Tong Yang1,2, Xin Ye1, Huan-Xin Jiang2,4, Jing-Hao Huang2,4,5 and Li-Song Chen1,2,6,7* Abstract Background: Boron (B)-toxicity is an important disorder in agricultural regions across the world Seedlings of ‘Sour pummelo’ (Citrus grandis) and ‘Xuegan’ (Citrus sinensis) were fertigated every other day until drip with 10 μM (control) or 400 μM (B-toxic) H3BO3 in a complete nutrient solution for 15 weeks The aims of this study were to elucidate the adaptive mechanisms of citrus plants to B-toxicity and to identify B-tolerant genes Results: B-toxicity-induced changes in seedlings growth, leaf CO2 assimilation, pigments, total soluble protein, malondialdehyde (MDA) and phosphorus were less pronounced in C sinensis than in C grandis B concentration was higher in B-toxic C sinensis leaves than in B-toxic C grandis ones Here we successfully used cDNA-AFLP to isolate 67 up-regulated and 65 down-regulated transcript-derived fragments (TDFs) from B-toxic C grandis leaves, whilst only 31 up-regulated and 37 down-regulated TDFs from B-toxic C sinensis ones, demonstrating that gene expression is less affected in B-toxic C sinensis leaves than in B-toxic C grandis ones These differentially expressed TDFs were related to signal transduction, carbohydrate and energy metabolism, nucleic acid metabolism, protein and amino acid metabolism, lipid metabolism, cell wall and cytoskeleton modification, stress responses and cell transport The higher B-tolerance of C sinensis might be related to the findings that B-toxic C sinensis leaves had higher expression levels of genes involved in photosynthesis, which might contribute to the higher photosyntheis and light utilization and less excess light energy, and in reactive oxygen species (ROS) scavenging compared to B-toxic C grandis leaves, thus preventing them from photo-oxidative damage In addition, B-toxicity-induced alteration in the expression levels of genes encoding inorganic pyrophosphatase 1, AT4G01850 and methionine synthase differed between the two species, which might play a role in the B-tolerance of C sinensis Conclusions: C sinensis leaves could tolerate higher level of B than C grandis ones, thus improving the B-tolerance of C sinensis plants Our findings reveal some novel mechanisms on the tolerance of plants to B-toxicity at the gene expression level Keywords: Boron-tolerance, Boron-toxicity, cDNA-AFLP, Citrus grandis, Citrus sinensis, Photosynthesis Background Althought boron (B) is a micronutrient element required for normal growth and development of higher plants, it is harmful to plants when present in excess Whilst of lesser importance than B-deficiency (a widespread problem in many agricultural crops), B-toxicity is also an important problem in agricultural regions across the world, which citrus trees are cultivated [1-3] Despite the * Correspondence: lisongchen2002@hotmail.com College of Resource and Environmental Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China Institute of Horticultural Plant Physiology, Biochemistry and Molecular Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, China Full list of author information is available at the end of the article importance of B-toxicity for crop productivity, the mechanisms by which plants respond to B-toxicity are poorly understood yet Recently, increasing attention has been paid to plant B-toxicity as a result of the increased demand for desalinated water, in which the B level may be too high for healthy irrigation of crops [4] Alteration of gene expression levels is an inevitable process of plants responding to environmental stresses Kasajima and Fujiwara first investigated high B-induced changes in gene expression in Arabidopsis thaliana roots and rosette leaves using microarray, and identified a number of high B-induced genes, including a heat shock protein and a number of the multi-drug and toxic compound extrusion (MATE) family transporters [5] Hassan et al © 2014 Guo et al.; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Guo et al BMC Plant Biology 2014, 14:284 http://www.biomedcentral.com/1471-2229/14/284 preformed suppression subtractive hybridization on root cDNA from bulked B-tolerant and -intolerant doubled haploid barley lines grown under moderate B-stress and identified 111 upregulated clones in the tolerant bulk under B-stress, nine of which were genetically mapped to B-tolerant quantitative trait loci An antioxidative response mechanism was suggested to provide an advantage in tolerating high level of soil B [6] Recently, Aquea et al found that B-toxicity upregulated the expression of genes related to ABA signaling, ABA response and cell wall modification, and downregulated the expression of genes involved in water transporters in Arabidopsis roots, concluding that root growth inhibition was caused by B-toxicity-induced water-stress [7] Most research, however, has focused on roots and herbaceous plants (i.e., barley, A thaliana), very little is known about the differential expression of genes in response to B-toxicity in leaves and woody plants Citrus belongs to evergreen subtropical fruit trees In China, B-toxicity often occurs in citrus orchards from high level of B in soils and/or irrigation water and from inappropriate application of B fertilizer especially under low-rainfall conditions [8,9] During 1998–1999, Huang et al investigated the nutrient status of soils and leaves from 200 ‘Guanximiyou’ pummelo (Citrus grandis) orchards located in Pinghe, Zhangzhou, China Up to 61.5% and 17.0% of orchards were excess in leaf B and soil water-soluble B, respectively [10] Previous studies showed that B-toxicity disturbed citrus plant growth and metabolism in multiple way, including interference of nutrient uptake [2], ultrastructural damage of roots and leaves [11-13], inhibition of CO2 assimilation, photosynthetic enzymes and photosynthetic electron transport, decrease of chlorophyll (Chl), carotenoid (Car) and total soluble protein levels, affecting leaf carbohydrate metabolism and antioxidant system [9,14] However, our understanding of the molecular mechanisms underlying these processes in citrus is very limited To our best knowledge, no high B-toxicity-induced changes in gene expression profiles have been reported in citrus plants to date Here we investigated the effects of B-toxicity on growth, leaf CO2 assimilation, leaf concentrations of malondialdehyde (MDA), pigments and total soluble protein, root and leaf concentration of B, leaf concentration of phosphorus (P), and leaf gene expression profiles using cDNA-amplified fragment length polymorphism (cDNA-AFLP) in Citrus grandis and Citrus sinensis seedlings differing in B-tolerance [13] The aims of this study were to elucidate the adaptive mechanisms of citrus plants to B-toxicity and to identify B-tolerant genes Results Effects of B-toxicity on seedlings growth, B concentration in roots and leaves, and P concentration in leaves Because B is phloem immobile in citrus plants, B-toxic symptoms first developed in old leaves The typical Page of 22 visible symptom produced in B-toxic leaves was leaf burn (chlorotic and/or necrotic), which only occurred in C grandis plants In the later stages, B-toxic leaves shed premature By contrast, almost no visible symptoms occurred in C sinensis plants except for very few plants (Additional file 1) B-toxicity-induced decreases in root, shoot and whole plant dry weights (DWs) were more pronounced in C grandis than in C sinensis seedlings (Figure 1A-C) Root DW decreased to a larger extent than shoot DW in response to B-toxicity, and resulted in a decrease in root DW/shoot DW ratio of both C grandis and C sinensis seedlings (Figure 1A-B and D) B-toxicity increased B concentration in roots and leaves, especially in leaves and decreased P concentration in C grandis leaves No significant differences were found in root and leaf B concentration and leaf P concentration between the two species at each given B treatment except that B concentration was higher in B-toxic C sinensis leaves than in B-toxic C grandis ones (Figure 2) Effects of B-toxicity on leaf gas exchange, pigments, total soluble protein and MDA B-toxicity-induced decreases in both CO2 assimilation and stomatal conductance were higher in C grandis than in C sinensis leaves Intercellular CO2 concentration increased in C grandis leaves, but did not significantly change in C sinensis leaves in response to B-toxicity CO2 assimilation and stomatal conductance in control leaves did not differ between the two species, but were higher in B-toxic C sinensis leaves than in B-toxic C grandis ones Intercellular CO2 concentration in control leaves was higher in C sinensis than in C grandis, but the reverse was the case in B-toxic leaves (Figure 3A-C) B-toxicity decreased concentrations of Chl a + b and Car and ratio of Chl a/b in C grandis and C sinensis leaves In control leaves, all the three parameters did not differ between the two species, but Chl a + b and Car concentrations were higher in B-toxic C sinensis leaves than in B-toxic C grandis ones (Figure 3E-G) Leaf concentrations of total soluble protein and MDA were decreased and increased by B-toxicity in C grandis leaves, respectively, but were not significantly affected in C sinensis ones (Figure 3D and H) B-toxicity-induced differentially expressed genes revealed by cDNA-AFLP Here we used a total of 256 selective primer combinations to isolate the differentially expressed transcript-derived fragments (TDFs) from B-toxic leaves of two citrus species differing in B-tolerance A representative picture of a silver-stained cDNA-AFLP gel showing B-toxicity-induced genes in C grandis and C sinensis leaves was presented in Additional file As shown in Table 1, a total of 6050 clear Guo et al BMC Plant Biology 2014, 14:284 http://www.biomedcentral.com/1471-2229/14/284 -1 a a 45 a b 10 b b c d 15 D B a 40 a b 30 30 c c b 20 0.4 b c 0.2 10 -1 60 C Root + shoot DW (g plant ) 15 Shoot DW (g plant ) C sinensis C grandis A Root DW/shoot DW -1 Root DW (g plant ) 20 Page of 22 0.0 Control B-toxicity Control B-toxicity Figure Effects of B-toxicity on growth of Citrus sinensis and C grandis seedlings Bars represent means ± SE (n =10) (A-C) Root, shoot and root + shoot DWs (D) Ratio of root DW to shoot DW Bars represent means ± SE (n =10) Different letters above the bars indicate a significant difference at P